Plasma complement component C2: a potential biomarker for predicting abdominal aortic aneurysm related complications

Blood-based adjunctive measures that can reliably predict abdominal aortic aneurysm (AAA)-related complications hold promise for mitigating the AAA disease burden. In this pilot study, we sought to evaluate the prognostic performance of complement factors in predicting AAA-related clinical outcomes. We recruited consecutive AAA patients (n = 75) and non-AAA patients (n = 75) presenting to St. Michael’s Hospital. Plasma levels of complement proteins were assessed at baseline, as well as prospectively measured regularly over a period of 2 years. The primary outcome was the incidence of rapidly progressing AAA (i.e. aortic expansion), defined as change in AAA diameter by either 0.5 cm in 6 months, or 1 cm in 12 months. Secondary outcomes included incidence of major adverse aortic events (MAAE) and major adverse cardiovascular events (MACE). All study outcomes (AAA diameter, MACE and MAAE) were obtained during follow-up. Multivariable adjusted Cox regression analyses were performed to assess the prognostic value of plasma C2 levels in patients with AAA regarding rapid aortic expansion and MAAE and MACE. Event-free survival rates of both groups were also compared. Compared to non-AAA patients, patients with AAA demonstrated significantly higher plasma concentrations of C1q, C4, Factor B, Factor H and Factor D, and significantly lower plasma concentrations of C2, C3, and C4b (p = 0.001). After a median of 24 months from initial baseline measurements, C2 was determined as the strongest predictor of rapid aortic expansion (HR 0.10, p = 0.040), MAAE (HR 0.09, p = 0.001) and MACE (HR 0.14, p = 0.011). Based on the data from the survival analysis, higher levels of C2 at admission in patients with AAA predicted greater risk for rapid aortic expansion and MAAE (not MACE). Plasma C2 has the potential to be a biomarker for predicting rapid aortic expansion, MAAE, and the eventual need for an aortic intervention in AAA patients.

The manufacturer's protocol was followed for the multiplex bead assays. Sample intra-assay Coefficients of Variability (CV) was < 10% while the inter-assay CV was 15%. Prior to any sample analysis, Fluidics Verification and Calibration bead kits (Luminex Corp) were used to calibrate the MagPix analyzer (Luminex Corp; Austin, Texas). At least 50 beads for each protein were acquired using Luminex xPonent software and analyzed using Milliplex Analyst software (v.5.1; EMD-Millipore).

Measured outcomes.
The primary outcome of this study was the rapid expansion of the AAA diameter observed during the follow-up period and defined as AAA size > 1 cm over 12 months or 0.5 cm over 6 months 3 . Secondary outcomes included the incidence of major adverse aortic events (MAAE) and major adverse cardiovascular events (MACE). MAAE was defined as the composite incidence of elective AAA repair (open or endovascular repair), emergent AAA repair, AAA-related deaths, and AAA-induced complication (arterial thrombosis due to emboli from AAA, primary aorto-enteric fistula or primary aortocaval fistulas). MACE was defined as the composite incidence of cardiovascular-related mortality, stroke, or myocardial infarction.
Two-year prospective follow-up. Over a period of 24 months after the initial baseline visit, patients were seen at 6-month or 12-month intervals (depending on their AAA size). This follow-up period was based on the AAA surveillance protocol recommended by the SVS guidelines 3 . During these follow-up visits, changes in clinical history or medications were recorded, AAA diameter was re-measured (via ultrasound), and the incidence of emergent AAA repair (secondary to the development of symptomatic AAA) or ruptured AAA were noted. Furthermore, the need for elective AAA repair (i.e. repair of AAA as per SVS guidelines-AAA diameter > 5 cm for females or > 5.5 cm for males) or repair of AAA due to rapid expansion were also recorded 3 . Baseline  www.nature.com/scientificreports/ plasma complement factor levels were assessed by the Kolmogorov-Smirnov test, and summarized as medians and interquartile ranges (IQRs) accordingly. Event rates for rapid AAA diameter expansion, MAAE, and MACE at 2 years were reported for the overall cohort and compared between AAA and non-AAA patient groups using chi-square test. Hazard ratios (HRs) and 95% confidence intervals (95% CIs) for events per one unit increase in plasma complement factors were calculated using univariable and multivariable models adjusted for age, sex, hypertension, dyslipidemia, smoking, and history of coronary artery disease. Receiver operator curve (ROC) analysis was conducted to identify a cut-off value for C2 that could facilitate stratification of AAA patients atrisk of adverse clinical outcomes into low versus high-risk groups. The cut-off value was chosen based on a high positive likelihood ratio (LR +) yielding a sensitivity above 90%. Overall event-free survival rates of both groups were displayed using Kaplan-Meier curves, and differences between curves were compared with a log-rank test.

Statistical analysis.
Significance was set at a two-tailed p < 0.05. All analyses were carried out using SPSS software version 23 (SPSS Inc., Chicago, Illinois, USA).

Ethics statement. The studies involving human participants were reviewed and approved by Unity Health
Toronto's Research Ethics Board. The patients/participants provided their written informed consent prior to participating in this study.

Results
Clinical characteristics. Baselines Table 4). On the other hand, we noted that few of the investigated proteins were able to predict some but not all investigated outcomes. An increase in plasma Factor H (per ug/mL) was associated with an increase in the risk of MAAE (adjusted HR 1.12  Correlation of plasma C2 levels and clinical variables. Among AAA and non-AAA patients, C2 plasma levels were not associated with any known established risk factor (age, sex, hypertension, hypercholesterolemia, diabetes, renal insufficiency, smoking, history of congestive heart failure, history of coronary artery disease, and history of stroke, p-value < 0.05) (Table 5). Similarly, C2 plasma levels were not associated with commonly used medications used to treat the listed risk factors (ACE inhibitors/ARBS, Aspirin, beta blockers, CCB, HCTZ, insulin, oral hypoglycemics, and statins, p-value < 0.05) ( Prognostication of study outcomes based on C2 levels at presentation. Based on the ROC curve, we identified a C2 concentration of 0.202 ug/mL (AUC of 0.709 (p = 0.010, 95% CI 0.641-0.763), likelihood ratio (LR) + 5.34, 91% sensitive, and 62% specific) as the optimal cutoff value to facilitate stratification of AAA patients at-risk of clinical complications into low-risk vs high-risk groups. Utilizing this cutoff value, AAA patients (n = 75) were divided into 2 groups, (1) Low C2 group (n = 24) with C2 concentration < 0.202 ug/mL and (2) High C2 group (n = 51) with C2 concentration > 0.202 ug/mL. The clinical characteristics of both these groups are highlighted in Table 6. Among AAA patients, a higher rate of rapid aortic expansion (33% vs. 4%, p = 0.001) and MAAE (67% vs. 33%, p = 0.007) was noted in patients with low C2 levels compared to the High C2 group. No significant difference was noted in the incidence of MACE (33% vs. 18%, p = 0.130) among AAA patients with high versus low C2 levels.

Discussion
In this study, we demonstrated significant differences in plasma levels of component factors in AAA patients versus non-AAA patients. Our analysis demonstrated that baseline C2 was a reliable predictor of all three measured outcomes in this study, including rapid aortic expansion, MAAE and MACE over a two-year follow-up period. Based on Kaplan-Meier analysis data, measuring C2 levels at baseline may aid and serve as a potential biomarker for stratifying patients at risk of rapid aortic expansion or MAAE (Fig. 2).
As various elements of the complement system are found in different strata of the aortic tissue, a growing body of evidence demonstrates the active involvement of the complement system in acute cardiovascular events and aortic disease [13][14][15] . In murine animals, complement C3a and C5a depletion were protective against AAA formation 13 . In contrast, Zagrapan et al. found significantly increased levels of circulating C5a factor in the Additionally, we demonstrated an association between low circulating C2 levels and increased risk for rapid aortic expansion and MAAE in patients with AAA. In comparison, Hinterseher et al. demonstrated an increase in gene expression of C1Qa, C1Q and C2 and a decrease in expression of C2 inhibitor SERPING1 in human aortic aneurysmal tissue 18 . Furthermore, they noted an increase in complement protein C2 staining in cells of aortic aneurysmal tissue 18 .
The pathogenesis behind our findings (i.e. the association between low levels of C2 in plasma and an increased expression of C2) within aneurysmal aortic tissue still needs to be investigated further. Previously, the Classical pathway has been shown to be independently activated by pentraxins, such as C-reactive protein (CRP), which has also been linked to aortic expansion in patients with AAA 19,20 . Homozygous C2 deficiency, in addition to its association with severe infections and rheumatic disease, has also been linked with various forms of vasculitis with cutaneous and gastrointestinal manifestations 21,22 . However, a direct link between C2 deficiency and Table 4. Multivariable Cox proportional hazards model assessing the association between complement factors and rapid expansion in AAA (ΔAAA size > 1 cm/12 months or 0.5 cm/6 months), MAAE and MACE, adjusted for age, sex, smoking, and History of CAD, p-value < 0.05. AAA , abdominal aortic aneurysm; MAAE, major adverse aortic event; MACE major adverse cardiac event; HR hazard ratio; CI confidence interval. www.nature.com/scientificreports/ aneurysm formation is yet to be established. Thus, the biological role of C2 in aortic aneurysm progression would undoubtedly be an area of interest that would warrant further investigation. To date, numerous circulating biomarkers have been investigated as potential predictive factors for AAA expansion and rupture. These markers can be categorized into those involved in the coagulation pathway 23-30 , extracellular matrix turnover and matrix degrading enzymes 23,30-42 and lipids 25,[43][44][45][46] . Furthermore, there have been various circulating biomarkers involved in the immune response system that have been investigated for an association with AAA expansion and/or rupture, which include, CRP 24,25,35,[46][47][48][49] , interleukin-1β 50 , interleukin-2 50 , interleukin-6 46,50,51 , interleukin-8 50 , interferon-gamma 52 , leukocytes 24 , macrophage inhibiting factor 23,53 , neutrophil gelatinase-associated lipocalin 54 , osteopontin 55 , osteoprotegerin 56 , peroxiredoxin 57 , tumour necrosis factor-α 46,50 , tumour necrosis factor-like weak inducer of apoptosis 58 and C5a 16 . The lack of data on the role of the complement system in aortic expansion led to further analysis of the relationship between circulating complement factors and aortic expansion. Table 5. Association between C2 levels and demographic data, cardiovascular risk factors and medications in 150 patients diagnosed with AAA and those without AAA. Continuous variables are shown in mean (standard deviation). ‡Data summarized as medians and interquartile ranges (IQRs). *Represents significant difference between AAA and no AAA patients; p < 0.05; differences between groups were compared using chi-square test. All p-values were rounded to three decimal places. AAA , abdominal aortic aneurysm.  59 . Furthermore, recent studies have cast doubt over whether the maximum diameter alone should guide the treatment of patients with AAA 60 . Notably, circulating biomarkers have also been greatly emphasized due to their capacity to provide important prognostic information about subsequent aortic behaviour, thereby allowing for more patient-specific management 8,48,[61][62][63][64] . At the time of writing this paper, more accurate prognostic predictors are needed to guide stratifying patients into those at risk for rupture rather than relying on diameter alone, as some small AAA are known to rupture, while some large AAAs can remain dormant for some time 4,65 . The current SVS guidelines suggest surveillance imaging for AAAs measuring 3.0-3.9 cm, 4.0-4.9 cm and 5.0-5.4 cm at 3-year, 12-month and 6-month intervals, respectively 3 . In contrast, our findings indicate that there may be a subgroup of AAA patients (those with low circulating plasma C2 at higher risk of rapid aortic expansion and MAAE) who may benefit from careful oversight and more frequent follow-up. Furthermore, circulating C2 levels may be utilized as a part of the clinical decision-making process to help reduce the risks associated with AAA treatment, particularly in high-operative-risk patients, until the risk of rupture is believed to outweigh the operative risk 4 . While our findings regarding plasma C2 levels may add to the potential biomarkers that can be used to prognosticate patients with AAA, further validation in a larger and more heterogeneous patient cohort is still required.
Limitations include the single-center nature of our study and the unaccounted study outcomes in patients lost to follow-up. A larger and more diverse sample size with prolonged follow-up may prove insightful in evaluating the true prognostication potential of C2 in patients with AAA, as this was a pilot study to determine whether the role of complement factors in AAA disease warrants further investigation. Future studies investigating the biological role of C2 in aneurysmal aortic tissue are also warranted. Not all complement factors and Table 6. Comparison of the Clinical Characteristics for AAA Patients with high and low plasma levels of C2. Continuous variables are shown by mean (standard deviation). ‡ Frequencies and percentages were calculated for categorical variables; α Event rate variables are shown in number (precent). *Represents significant difference between Low C2 and High C2 patients; p < 0.05; differences between groups were compared using chi-square test. All p-values were rounded to three decimal places. AAA abdominal aortic aneurysm; MAAE major adverse aortic event; MACE major adverse cardiac event.  www.nature.com/scientificreports/ their associated activated forms and substrates were investigated in this trial. Lastly, there may have additional confounding factors aside from the ones measured in this study that may correlate with plasma C2 levels, which will surely need to be examined in future studies.
In conclusion, we demonstrated that C2 has a strong predictive potential for AAA-related complications despite adjusting for confounding factors. Provided our findings are validated, circulating plasma C2 may be used in the future as a viable adjunct blood-based biomarker for the identification of AAA patients at high risk of rapid expansion and MAAE.

Data availability
All data generated or analyzed during this study are included in this published article.